Prestalk-like positioning of de-differentiated cells in the social amoeba Dictyostelium discoideum

The social amoeba Dictyostelium discoideum switches between solitary growth and social fruitification depending on nutrient availability. Under starvation, cells aggregate and form fruiting bodies consisting of spores and altruistic stalk cells. Once cells socially committed, they complete fruitification, even if a new source of nutrients becomes available. This social commitment is puzzling because it hinders individual cells from resuming solitary growth quickly. One idea posits that traits that facilitate premature de-commitment are hindered from being selected. We studied outcomes of the premature de-commitment through forced refeeding. Our results show that when refed cells interacted with non-refed cells, some of them became solitary, whereas a fraction was redirected to the altruistic stalk, regardless of their original fate. The refed cells exhibited reduced cohesiveness and were sorted out during morphogenesis. Our findings provide an insight into a division of labor of the social amoeba, in which less cohesive individuals become altruists.

fragment was obtained from the pspA-Gal construct (Dicty Stock Center plasmid ID 49) 3

by excision at
XbaI and BglII sites.
Strains constitutively expressing GFP or RFP were constructed by transforming AX4 with GFP or RFP expression constructs under the control of the act15 promoter, with 10 μg/mL G418 selection.

(b) Refeeding experiments
Vegetative cells were washed with phosphate buffer (PB) (pH 6.5; 20 mM KH2PO4, 20 mM Na2HPO4) and plated on a 1% PB agar (BactoAgar, Difco) plate at 0.3 to 0.4×10 6 cells/cm 2 and incubated at 22°C for 18 h until the slug stage.The cells were mechanically dissociated in PB containing 20 mM EDTA (pH 6.4) through repeated pipetting and passage through a 23 G needle and a 40μm cell strainer, followed by washing with PB.
To prepare a bacterial suspension, E. coli B/r was grown in LB medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) until it reached an optical density (OD) of 3 at 600 nm.The culture was concentrated in PB at OD50 equivalent by centrifugation.The bacterial suspension was immediately used.
For refeeding, dissociated cells were co-suspended with E. coli B/r at OD50 in PB at a final density of 2 to 3×10 6 cells/ml.The mixture was shaken for 3 or 5 h in a 50 ml centrifuge tube with a typical volume of 1.25 mL.The cells were then washed by pelleting and resuspending in PB.This washing procedure was repeated twice to remove bacteria.In the case of refeeding with growth media, the PS growth medium was used.NF cells were cells suspended in PB without nutrients immediately after dissociation.
For the mixing experiments (Figs. 1 and 2), the NF and RF cells were mixed in an 85:15 ratio.
The mixed cell suspension adjusted to 1 to 2×10 6 cells/mL was plated on a glass-bottomed dish (MatTek, Ashland, MA) covered with a thin sheet of 1% PB agar.The plates were left undisturbed for 15 min to allow the cells to attach to the agar before gently removing the buffer.
Confocal images of GFP, RFP, and calcofluor white fluorescence were captured using an inverted confocal microscope (A1+, Nikon), and Z-slices were obtained using Ti Z-drive.The cells were observed at three time windows after plating: 1.5-2 h for the aggregates with a 20× air objective lens, 4-5 h during early culmination for the upper part of a fruiting body with a 40× air objective lens, 8-10 h for the basal disk of terminally differentiated fruiting bodies with a 60× oil immersion objective lens, and for cells scattered around the base of fruiting bodies with a 20× air objective lens.
Images were analyzed using custom programs in ImageJ 6 and the R language 7 .To quantify cell localization, images were binarized according to the fluorescence of the cell fate markers.Lists of XY coordinates of the marker-positive pixels were obtained from the binarized images.For the analysis of cell aggregates, Z-sections were taken at 3 µm intervals between 30 to 60 µm from the bottom of the aggregate.The XY coordinates of each Z-slice were merged into a single list.The origin of the coordinates was set to the aggregate center, and the aggregate radius was normalized to 1.The average distance from the center to the marker-positive pixels was computed for the prestalk and prespore markers.
For the analysis of early culminants, the upper regions of fruiting bodies were attached to a glass slide, and Z-slices were acquired at 5 µm intervals from the bottom to the top of the sample.The lists of XY coordinates of marker-positive pixels from the Z-slices were merged.Each fruiting body was vertically aligned from the tip to the lower cup, and a normalized coordinate in the vertical direction was assigned from 0 to 1.The frequency of marker-positive pixels at each position was quantified for the prestalk and prespore markers.The vertical position was divided into 50 equally-spaced bins to obtain histograms.
For the analysis of the basal disk, stalks were transferred to a glass slide and stained with 0.01% calcofluor white (BD, NJ, USA).Basal disk regions were determined using calcofluor white fluorescence which stains cellulose from vacuolated stalk cells 8 .Z-section images were acquired at a 1.5µm interval from the bottom to the top of the sample.The number of marker-positive pixels in the region was acquired from binarized images, and the values from each slice were summed.The ratio of the pixel numbers between the prestalk and prespore images was calculated.
For cells scattered around the base of fruiting bodies, the fruiting body standing on an agarcoated glass-bottom dish was observed from the bottom of the dish.Z-stack images were captured from the bottom of the sample to a height of 30 µm with 5 µm intervals.The number of markerpositive pixels was acquired within a 100 µm radius from the center of the basal disk, and the values from each Z-slice were summed.Ratios between the values obtained from the prestalk and prespore images were calculated.

(c) Quantitative real-time polymerase chain reaction analysis
qRT-PCR analysis was performed as follows: For 'No-dissociation' sample (Fig. 1D), AX4 vegetative cells were starved on a 1% PB agar plate and harvested at the selected time points.For the other conditions ('No-nutrient buffer', 'Bacteria', 'Growth medium' in Fig. 1D), dissociated slug cells were either suspended in PB or in PB mixed with E. coli at OD50 or in the PS growth medium.The suspension was shaken in a 50 ml centrifuge tube.Cells were harvested at the selected time points.Total RNA was extracted using a Maxwell 16 LEV simplyRNA cells and Tissue kit (Promega, WI, USA).cDNA was synthesized using random hexamers and SuperScript III (First-Strand Synthesis, Invitrogen).
Quantitative PCR (qPCR) amplification was performed with a qPCR thermocycler (ABI7500, Applied Biosystems) using a pre-mixed reaction solution (TaqMan Universal PCR Master Mix, Applied Biosystems) and with primer pairs and fluorescent beacons (TaqMan probe MGB, Applied Biosystems) (Table S2).The amplification value at the threshold cycle (CT) of each sample was measured in three independent wells, and the average value was used.The levels of relative gene expression were calculated from the CT and the relative standard curves for each gene, followed by normalization using rnlA amplification as an endogenous control.
For hierarchical clustering of time series of the gene expression level, permutation distribution clustering was applied with R function 'pdc'.Time series from the three conditions ('No-nutrient buffer', 'Bacteria', and 'Growth medium') were embedded and the squared Hellinger distance was calculated to measure dissimilarity between gene targets.Subsequently, clustering was performed based on the dissimilarity.The results are presented in Fig. 1D.
To investigate temporal differences in gene expression levels, 'hypothesis testing with bootstrap' 9 was applied to the qRT-PCR data.Pair-wise comparisons were performed between the sampling time points 0h and 5h.The null hypothesis was that both the samples originated from the same probability distribution.We generated pairs of 100,000 bootstrap samples from a mixture of the observed data.The percentile rank of the observed t-values within the distribution of t-values was determined through the bootstrap sampling.And then, the percentile rank was used as the Pvalue for a two-sided test.The results are presented in Table S1.

(d) Cell cohesiveness assay
Cell cohesiveness was assayed as previously described 10 with minor modifications to the cell density and container geometry.Dissociated slugs with the cell fate markers were suspended at 6×10 6 cells/mL.The suspensions were placed in 50 mL tubes and shaken at 120 rpm for 1.5, 3, and 5 h.In the NF condition, cells were suspended in plain PB.In RF conditions, cells were suspended in PB with E. coli at OD50 or the PS growth medium.To quantify the number of cells not associated with aggregates, images of the samples loaded into a hemocytometer were acquired and binarized based on the fluorescence of the cell fate markers.The number of single cells was then calculated using the particle analyzer function in ImageJ.The results are presented in Fig. 1E.

(e) Flow cytometry
Dissociated cells with the cell fate markers were suspended in PB mixed with E. coli B/r at OD50 and shaken for 3, 6, and 12 h.The cells were washed with PB, and the intensity of RFP and GFP expression was measured using a flow cytometer (SH800, Sony, Japan).Cells that were negative for ecmAO-RFP fluorescence and positive for pspA-GFP fluorescence were identified as 'Prespore fate', whereas cells that were positive for ecmAO-RFP fluorescence and negative for pspA-GFP fluorescence were categorized as 'Prestalk fate'.Cells that were negative for both ecmAO-RFP and pspA-GFP fluorescence were identified as 'Non-fluorescent cells'.The results are presented in Fig. S5.

(f) Terminal cell fate allocation within RF and NF cell mixture
In the terminal fate (spores and solitary cells), we quantified the ratio of RF cells to NF cells.For NF and RF cells, we utilized cells expressing GFP or RFP constitutively under the strong actin15 promoter.
The NF and RF cells were mixed at 1:1 ratio.The cell mixtures were applied on a 1% PB agar plate.
After incubating at 22°C for 10 h, when the development of the fruiting body had more or less completed, PB containing 20 mM EDTA was poured onto the agar plate, and spores and solitary amoeboid cells were harvested by pipetting.The cell suspension was treated with 0.01% calcofluor white (BD, NJ, USA) to distinguish spores from amoeboid cells 8 .The identification of RF cells from NF cells is based on the expression of GFP and RFP.Images of the samples loaded into a hemocytometer were acquired, and binarized based on the fluorescence, and the number of cells was calculated using the particle analyzer function in ImageJ.For the illustration in Fig. S4A, the GFP-RFP ratio in the terminal state was adjusted using the ratio of GFP-RFP in cells immediately after mixing, and then normalized by the control mixture NF(GFP)/NF(RFP).Additionally, Fig. S4B presents the data without adjustment and normalization for interpretation.

(h) Statistical analysis
All statistical analyses were performed using the R software 7 .Details of a generalized linear model (GLM) and analysis of deviance for the fit are listed in Table S1.For multiple comparisons, the P-value was adjusted using Holm's method.respectively.Prestalk cells were found in the peripheral region of the aggregate, while prespore cells were more uniformly distributed.Furthermore, RF cells formed a mound and tip (arrow) at roughly the same time as NF cells.These results indicate that RF cells were still fully capable of developing on their own.Therefore, the segregation between NF and RF cells in aggregates was not due to the innate inability of RF cells to develop, but rather a consequence of their relative behavior when in association with the NF cells.Lower panels: RF3+NF.The numbers in the images are the height from the bottom of an aggregate (µm).In the NF+NF condition, prestalk cells localized to the bottom of the aggregate, while prespore cells were found in the upper regions.In the RF3+NF condition, RF cells of both prestalk and prespore origins localized to the bottom.

(B)
The time series of fruiting body formation.The maximum intensity projections of Z-stack images are shown.In the NF+NF condition, NF prespore cells occupied the middle region of the fruiting body (arrows).In the RF3+NF condition, RF cells localized to the lower cup region of the fruiting body.
These results indicate that RF prespore cells lost their ability to occupy the upper region of the fruiting body.The numbers on the upper right side represent hour:min, with 0:00 indicating the starting point of imaging during the aggregation phase.The time points were chosen to highlight significant cell dynamics, such as culmination and cell positioning.For a comprehensive view of the dynamics, see Movie S3 and S4.The quantification of cell positioning (Fig. 2B) was based on fruiting bodies observed 4-5h after plating, consistent across both the NF+NF and RF+NF conditions.All scale bars = 50 µm.Additionally, although strains constitutively expressing GFP or RFP were utilized, there was a tendency for slightly less basal production of spores and solitary cells in the GFP strain.Despite these challenges, at least, the results indicate that both RF(GFP) and RF(RFP) exhibited proportional changes compared to NF cells.In the statistical analysis, the crude count data in the terminal state was used as response variable, and the ratio of cells with GFP toward RFP in cells of immediately after mixing was used as an offset term to adjust the response variable.In the RF+NF, RF cells were redirected into a higher proportion of solitary amoeboid cells than NF cells (GLMM and analysis of deviance, P < 0.0001, Table S1).On the other hand, NF cells differentiated into a higher proportion of spore than RF cells (GLMM and analysis of deviance, P < 0.0001, Table S1).Sample size N represents three biological replicates with at least 3600 cells (amoeboid cells and spores) per condition.

Model formula R code format Explanation
The continuation of Table S1.

Analysis Quantitative real-time polymerase chain reaction analysis for Fig. 1D
Analysis to detect temporal differences in the levels of gene expression Explanation Pair-wise comparisons of levels of relative gene expression were performed between the sampling time points 0h and 5h with the bootstrap method.For the detail, see SI text, 'Quantitative realtime polymerase chain reaction analysis'.

Results
Bootstrap The continuation of Table S1.

Results
To understand cell cohesion change incurred by refeeding, following comparisons are considered.

Results
To understand the effect of refeeding on prespore ratio in the unattached cells, the following comparisons are considered.GLM with gamma error.Rv: , Ev: .Link: () = ().: Prespore frequency at each vertical position in a fruiting body.: Prestalk frequency at the corresponding vertical position.To use log link function, we added 0.1 to the score of  and  before analysis, because these included a score 0.

Results
No-dissociation: slope =- The continuation of Table S1.

Analysis
Prespore ratio among cells left behind in basal region for Fig. 2G.

Explanation
Same as the model formula for "Prespore marker expression ratio in a basal disk for Fig. 2E".

Figure
Figure S1 The time course of the reaggregation of dissociated cells.Time-series of the development following the plating of cell mixtures (NF+NF or RF+NF) are presented, capturing images from the moment of plating (0:00) until the characteristic sorting pattern emerges.The quantification of cell positioning (Fig.1 B-C) was based on aggregations 1.5-2 hours after plating, consistent across both the NF+NF and RF+NF conditions.The merged fluorescent and bright field images.In the NF+NF condition, prestalk cells (ecmAO-RFP, magenta) were sorted to the peripheral region of the aggregate, whereas prespore cells (pspA-GFP, green) were more uniformly distributed.In the RF3+NF condition, RF cells of both prestalk and prespore origins were well mixed and subsequently sorted out to the periphery of the aggregates.The numbers on the upper left side indicate hour:min.All scale bars = 50 µm.

Figure
Figure S2 Supporting data for refeeding experiments.(A) The swap control.NF cells with the cell-type reporter (fluorescent) and nonfluorescent RF3 cells were mixed at a ratio 15:85 (hereafter referred to as RF(Fl-)+NF(Fl+)).Representative images of mixed aggregates (pspA: GFP-channel.ecmAO: RFP-channel.Merged: overlay of bright-field (grayscale) and fluorescence images).A reciprocal pattern of fluorescent cells in the center and non-fluorescent cells in the periphery were shown.The result indicates that expression of the marker genes itself did not affect the sorting pattern.(B) RF cells were plated alone on the non-nutrient agar and their aggregation was observed.'2h' and '3.5h' indicate the time after plating.'RF3 alone' and 'RF5 alone' indicate RF3 or RF5 cells plated alone,

( C )
Experiments to show the requirement of nutrient replenishment for the segregation.RF(Nonutrient)+NF: Cells with fluorescent markers were shaken for 3 h in the plain phosphate buffer, and mixed with nonfluorescent NF cells.The result indicates that cells shaken in the non-nutrient buffer were not segregated from the freshly dissociated cells.RF(growth medium)+NF: Cells with fluorescent were shaken for 3 h in the growth medium, and mixed with nonfluorescent NF cells.Refed cells with growth medium were sorted to the peripheral region, whereas NF cells were more uniformly distributed.The result suggests that the segregation pattern of refeeding with growth medium is the same as that of refeeding with bacteria.Taken together, these experiments indicate that the unique sorting pattern was not merely due to mechanical interruption but also required nutrient replenishment.All scale bars = 50 µm.

Figure
Figure S3 Fruiting body formation of refed cells.(A) Z stack images of an aggregate in the tip formation stage (3h after plating).Upper panels: NF+NF.

Figure S4
Figure S4 Terminal cell fate allocation in the mixed cell population.(A) GFP strain ratio in the cell mixtures with a normalization.NF and RF cells constitutively expressed GFP or RFP.The mixture of NF GFP cells and NF RFP cells (NF(GFP)/NF(RFP)) is the control mixture.Left panel: The mixtures of RF GFP cells with NF RFP cells, denoted as (RF3(GFP)/NF(RFP), RF5(GFP)/NF(RFP)).Right panel: The mixtures of RF RFP cells with NF GFP cells, denoted as (NF(GFP)/RF3(RFP), NF(GFP)/RF5(RFP)).For the illustration, the GFP ratio was adjusted using the ratio of GFP-RFP in cells immediately after mixing, and then normalized by the control mixture NF(GFP)/NF(RFP).Error bars: Standard error.(B) Cell ratio in the cell mixtures without the adjustment and normalization.The ratio represents the average of three biological replicates.Despite our strict control over experimental conditions, the experiments tend to exhibit slightly larger variation, likely due to the dissociation, refeeding, and initiation of growth in RF solitary cells.

Figure S5 Flow
Figure S5 Flow cytometry analysis of cells during refeeding.(A) Scatter plot showing fluorescent intensities of cell fate markers ecmAO-RFP (prestalk) and pspA-GFP (prespore).Cells from dissociated slugs were incubated in the bacterial suspension.(B) Temporal changes in the ratio of cell types.Error bars: 95% confidence interval (CI).

Table S1
Summary of statistical analysis.GLM is generalized linear model. !,  # and  $ indicate regression coefficients.Rv: Response variable, Ev: Explanatory variable.Link: Link functions that decide relationship between Rv and Ev.
* indicates P <0.05AnalysisCell localization within aggregations for Fig.1C <0.025.The p-value was the percentile rank of the observed t-values within bootstrap sampling data.For a two-sided test, P <0.025 was used for the significance.

Table S2
Primer pairs and fluorescent beacons for qRT-PCR.* indicates primers and beacons that are newly constructed in this study.The rest are previously described 11 .